Elspeth F. Garman

High resolution structures of HIV-1 RT from four RT-inhibitor complexes.

We have determined the structures of four complexes of HIV-1 reverse transcriptase with non-nucleoside inhibitors, three fully refined at high resolution. The highest resolution structure is of the RT-nevirapine complex which has an R-factor of 0.186 and a root-mean-square bond length deviation of 0.015 Å for all data to 2.2 Å. The structures reveal a common mode of binding for these chemically diverse compounds. The common features of binding are largely hydrophobic interactions and arise from induced shape complementarity achieved by conformational rearrangement of the enzyme and conformational/ conf igurational rearrangement of the compounds.

Architecture of a protein central to iron homeostasis : crystal structure and spectroscopic analysis of the ferric uptake regulator.

Iron is an essential element for almost all organisms, although an overload of this element results in toxicity because of the formation of hydroxyl radicals. Consequently, most living entities have developed sophisticated mechanisms to control their intracellular iron concentration. In many bacteria, including the opportunistic pathogen Pseudomonas aeruginosa, this task is performed by the ferric uptake regulator (Fur). Fur controls a wide variety of basic physiological processes including iron uptake systems and the expression of exotoxin A. Here, we present the first crystal structure of Fur from P. aeruginosa in complex with Zn2+ determined at a resolution of 1.8 Å. Furthermore, X‐ray absorption spectroscopic measurements and microPIXE analysis were performed in order to characterize the distinct zinc and iron binding sites in solution. The combination of these complementary techniques enables us to present a model for the activation and DNA binding of the Fur protein.

Haem-ligand switching during catalysis in crystals of a nitrogen-cycle enzyme.

Cytochrome cd1 nitrite reductase catalyses the conversion of nitrite to nitric oxide in the nitrogen cycle. The crystal structure of the oxidized enzyme shows that the d1 haem iron of the active site is ligated by His/Tyr side chains, and the c haem iron is ligated by a His/His ligand pair. Here we show that both haems undergo re-ligation during catalysis. Upon reduction, the tyrosine ligand of the d1 haem is released to allow substrate binding. Concomitantly, a refolding of the cytochrome c domain takes place, resulting in an unexpected change of the c haem iron coordination from His 17/His 69 to Met106/His69. This step is similar to the last steps in the folding of cytochrome c. The changes must affect the redox potential of the haems, and suggest a mechanism by which internal electron transfer is regulated. Structures of reaction intermediates show how nitric oxide is formed and expelled from the active-site iron, as well as how both haems return to their starting coordination. These results show how redox energy can be switched into conformational energy within a haem protein.

HDAC6–p97/VCP controlled polyubiquitin chain turnover

HDAC6 is a unique cytoplasmic deacetylase capable of interacting with ubiquitin. Using a combination of biophysical, biochemical and biological approaches, we have characterized the ubiquitin‐binding domain of HDAC6, named ZnF‐UBP, and investigated its biological functions. These studies show that the three Zn ion‐containing HDAC6 ZnF‐UBP domain presents the highest known affinity for ubiquitin monomers and mediates the ability of HDAC6 to negatively control the cellular polyubiquitin chain turnover. We further show that HDAC6‐interacting chaperone, p97/VCP, dissociates the HDAC6–ubiquitin complexes and counteracts the ability of HDAC6 to promote the accumulation of polyubiquitinated proteins. We propose that a finely tuned balance of HDAC6 and p97/VCP concentrations determines the fate of ubiquitinated misfolded proteins: p97/VCP would promote protein degradation and ubiquitin turnover, whereas HDAC6 would favour the accumulation of ubiquitinated protein aggregates and inclusion body formation.

Crystal structure of Vibrio cholerae neuraminidase reveals dual lectin-like domains in addition to the catalytic domain

BACKGROUND Vibrio cholerae neuraminidase is part of a mucinase complex which may function in pathogenesis by degrading the mucin layer of the gastrointestinal tract. The neuraminidase, which has been the target of extensive inhibitor studies, plays a subtle role in the pathology of the bacterium, by processing higher order gangliosides to GM1, the receptor for cholera toxin. RESULTS We report here the X-ray crystal structure of V. cholerae neuraminidase at 2.3 A resolution. The 83 kDa enzyme folds into three distinct domains. The central catalytic domain has the canonical neuraminidase beta-propeller fold, and is flanked by two domains which possess identical legume lectin-like topologies but without the usual metal-binding loops. The active site has many features in common with other viral and bacterial neuraminidases but, uniquely, has an essential Ca2+ ion which plays a crucial structural role. CONCLUSIONS The environment of the small intestine requires V. cholerae to secrete several adhesins, and it is known that its neuraminidase can bind to cell surfaces, and remain active. The unexpected lectin-like domains possibly mediate this attachment. These bacterial lectin folds represent additional members of a growing lectin superfamily.

The crystal structure of cyclin A.

BACKGROUND Eukaryotic cell cycle progression is regulated by cyclin dependent protein kinases (CDKs) whose activity is regulated by association with cyclins and by reversible phosphorylation. Cyclins also determine the subcellular location and substrate specificity of CDKs. Cyclins exhibit diverse sequences but all share homology over a region of approximately 100 amino acids, termed the cyclin box. From the determination of the structure of cyclin A, together with results from biochemical and genetic analyses, we can identify which parts of the cyclin molecular may contribute to cyclin A structure and function. RESULTS We have solved the crystal structure, at 2.0 A resolution, of an active recombinant fragment of bovine cyclin A, cyclin A-3, corresponding to residues 171-432 of human cyclin A. The cyclin box has an alpha-helical fold comprising five alpha helices. This fold is repeated in the C-terminal region, although this region shares negligible sequence similarity with the cyclin box. CONCLUSIONS Analysis of residues that are conserved throughout the A, B, and E cyclins identifies two exposed clusters of residues, one of which has recently been shown to be involved in the association with human CDK2. The second cluster may identify another site of cyclin A-protein interaction. Comparison of the structure of the unbound cyclin with the structure of cyclin A complexed with CDK2 reveals that cyclin A does not undergo any significant conformational changes on complex formation. Threading analysis shows that the cyclin-box fold is consistent with the sequences of the transcription factor TFIIB and other functionally related proteins. The structural results indicate a role for the cyclin-box fold both as a template for the cyclin family and as a generalised adaptor molecule in the regulation of transcription.

Radiation damage in macromolecular crystallography: what is it and why should we care?

The basic causes of the radiation damage inflicted on macromolecular crystals during diffraction experiments are summarized, as well as the current state of research which attempts to understand and to mitigate it.

Absorbed dose calculations for macromolecular crystals: improvements to RADDOSE

Radiation damage is an unwelcome and unavoidable aspect of macromolecular crystallography. In order to quantify the extent of X-ray-induced changes, knowledge of the dose (absorbed energy per unit mass) is necessary since it is the obvious metric against which to plot variables such as diffraction intensity loss and B factors. Significant improvements to the program RADDOSE for accurately calculating the dose absorbed by macromolecular crystals are presented here. Specifically, the probability of energy loss through the escape of fluorescent photons from de-excitation of an atom following photoelectric absorption is now included. For lighter elements, both the probability of fluorescence and of its subsequent escape from the crystal are negligible, but for heavier atoms the chance of fluorescence becomes significant (e.g. 30% as opposed to Auger electron decay from a K-shell excited iron atom), and this has the effect of reducing the absorbed dose. The effects of this phenomenon on dose calculations are presented for examples of crystals of an iron-containing protein, 2-selenomethionine proteins, a uranium derivatised protein, and for a nucleic acid sample. For instance, the inclusion of fluorescent escape results in up to a 27% decrease in the calculated absorbed dose for a typical selenomethionine protein crystal irradiated at the selenium K-edge.

Crystal structures of fibronectin-binding sites from Staphylococcus aureus FnBPA in complex with fibronectin domains.

Staphylococcus aureus can adhere to and invade endothelial cells by binding to the human protein fibronectin (Fn). FnBPA and FnBPB, cell wall-attached proteins from S. aureus, have multiple, intrinsically disordered, high-affinity binding repeats (FnBRs) for Fn. Here, 30 years after the first report of S. aureus/Fn interactions, we present four crystal structures that together comprise the structures of two complete FnBRs, each in complex with four of the N-terminal modules of Fn. Each ≈40-residue FnBR forms antiparallel strands along the triple-stranded β-sheets of four sequential F1 modules (2–5F1) with each FnBR/2–5F1 interface burying a total surface area of ≈4,300 Å2. The structures reveal the roles of residues conserved between S. aureus and Streptococcus pyogenes FnBRs and show that there are few linker residues between FnBRs. The ability to form large intermolecular interfaces with relatively few residues has been proposed to be a feature of disordered proteins, and S. aureus/Fn interactions provide an unusual illustration of this efficiency.

X-ray crystallographic structure of a papain-leupeptin complex.

The three‐dimensional structure of the papain‐leupeptin complex has been determined by X‐ray crystallography to a resolution of 2.1 Å (overall R‐factor = 19.8%). The structure indicates that: (i) leupeptin contacts the S subsites of the papain active site and not the Ssubsites; (ii) the ‘carbonyl’ carbon atom of the inhibitor is covalently bound by the Cys‐25 sulphur atom of papain and is tetrahedrally coordinated; (iii) the ‘carbonyl’ oxygen atom of the inhibitor faces the oxyanion hole and makes hydrogen bond contacts with Gln‐19 and Cys‐25.